The objective of this work is to develop and apply biochemical methods for the in vivo characterization of the transcription machinery in E. coli. Specifically, a novel primer-extension method will be used to probe the in vivo structure and the in vivo interactions of regulatory DNA of established nucleotide sequence. The close contacts made in vivo between several repressors and their operators will be determined. These will be compared to existing in vitro information if available, but will also be applied to a repressor which has not been purified. Basically, the aim is to obtain "footprints" and "alkylation-protection" patterns of regulatory DNA inside living E. coli. Another aim is to test current detailed models for operon-specific repression and for catabolite repression. The extent of repression will be adjusted in vivo and the quantitative occupancy of the putative DNA regulatory elements monitored by primer extension methods. Sites of proposed catabolite and operon-specific repression will be monitored to test whether their occupancy correlates quantitatively with repression, or whether alternative proposed mechanisms must be considered. A primer extension method will be developed which allows direct DNA sequence analysis of E. coli genomic DNA. This will be used to sequence mutations directly from E. coli DNA, bypassing the need for cloning. These experiments will provide basic information about normal and abnormal gene expression which is essential to learning the molecular basis of many disease states. In addition, the bypassing of lengthy cloning and purification of regulatory components and direct sequence analysis of genomic mutants should accelerate the rate of health research in general.